Wortmannin, an inhibitor of phosphatidylinositol kinases, blacks the MgATP-dependent recovery of Kir6.2/SUR2A channels

1. In order to investigate the mechanism underlying MgATP-dependent recovery of ATP-sensitive potassium (K-ATP) channels, we expressed Kir6.2/SUR2A (inwardly rectifying K+ channel subunit/sulfonylurea receptor) or C-terminal-truncated Kir6.2 (Kir6.2 Delta C26) in COS7 cells (Green monkey kidney cells), and carried out inside-out patch clamp experiments. 2. After patch excision in ATP-free internal solution, the activity of Kir6.2/SUR2A channels could be maximally recovered by the application of 5 mM MgATP. Subsequent application of 100 mu M Ca2+ induced a rapid decay of Kir6.2/SUR2A activity to 11.6 +/- 1.1% (mean +/- S.E.M) of the control level (Ca2+-induced run-down; n = 64). 3. MgATP (5 mM) recovered 99.4 +/- 4.2% (n = 13) of the C2+-induced run-down. Protein kinase inhibitors such as W-7, H-7, H-8 and genistein did not inhibit this reaction. However, wortmannin, an inhibitor of phosphatidylinositol 3- and 4-kinases, blocked the MgATP-dependent recovery in a concentration-dependent manner; the magnitudes of recovery were 35.7 +/- 7.2% (10 mu M) and 4.3 +/- 2.5% (100 mu M) of the Ca2+-induced run-down. 4. MgUDP (10 mns) reversed the Ca2+-induced run-down of KirB.2/SUR2A channels by 60.4 +/- 7.6% (n = 5). Wortmannin failed to modify this reaction. 5. Kir6.2 Delta C26 channels, which opened ill the absence of SUR2A, were less sensitive to Ca2+ Kir6.2 Delta C26 channels were inactivated to 44.8 +/- 4.4% (n =14) by 100 mu M Ca2+. MgATP recovered the Ca2+-induced run-down of Kir6.2 Delta C26 by 89.8 +/- 7.7 % (n = 9), and 100 mu M wortmannin inhibited this reaction (1.8 +/- 2%, n = 7). 6. Application of 10 mu M phosphatidylinositol-4,5-bisphosphate (PI-4,5-P-2) recovered the activity of Kir6.2/SUR2A channels after Ca2+-induced run-down (104.3 +/- 6.4%, n= 10). Even after the MgATP-dependent recovery was blocked by 100 mu M wortmannin, PI-4,5-P-2 reactivated the channels (102.3 +/- 8.6%, n=5). Similar results were obtained with Kir6.2 Delta C26. 7. These results suggest that the entity of MgATP-dependent recovery may be membrane Lipid phosphorylation rather than protein phosphorylation, and that synthesis of PI-4,5-P-2 or phosphatidylinositol-3,4,5-trisphosphate may upregulate Kir6.2 channels.